Avibactam and carbapenems antibacterial agents

ABSTRACT

Described are methods of treating or preventing a bacterial infection by administering an antibacterial agent comprising a β-lactamase inhibitor and one or more carbapenem to a subject.

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patentapplication 62/450,295 filed Jan. 25, 2017, which is hereby incorporatedby reference for all purposes as if fully set forth herein.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with government support under grant no.R21AI121805 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Mycobacterium abscessus is a rapidly growing non-tuberculousmycobacterium (NTM) found widely in soil and water and can cause aspectrum of infections. Prevalence of M. abscessus infections in thelungs of people with chronic conditions such as cystic fibrosis issignificant and can often lead to serious morbidity and mortality. Asurvey revealed that M. abscessus is present in the sputum of ˜13% ofcystic fibrosis patients in the US. Among NTM lung infections, M.abscessus is one of the prevalent species and often leads to a chronicand incurable disease. Drug resistance in M. abscessus is steadilyrising globally making it increasingly difficult to manage infectionswith these strains. Therefore, new drugs and novel regimens are acutelyneeded to treat infections with M. abscessus. An ideal new drug wouldinhibit a novel target so that it can be effective against M. abscessusstrains that are resistant to currently used drugs.

SUMMARY OF THE INVENTION

One embodiment of the present invention is a method of treating orpreventing a bacterial infection comprising the following steps:administering an agent comprising a β-lactamase inhibitor and one ormore carbapenem to a subject and treating or preventing a bacterialinfection in the subject. Examples of suitable carbapenems used in thepresent invention includes ertapenem, meropenem, imipenem, doripenem,biapenem, faropenem, tebipenem, panipenem and a combination thereof.Examples of suitable β-lactamase inhibitors used in the presentinvention includes is avibactam or clavulanate, or a combinationthereof. Within a method of the present invention the treating ofbacterial infection occurs when at least one symptom of the bacterialinfection is alleviated compared to a reference subject that was notbeen administered the agent. A preferred β-lactamase inhibitor isavibactam especially when treating a Mycobacterium abscessus infection.An example of a suitable amount of β-lactamase inhibitor that may beused in the present invention include an amount that is sufficient of toreduce the minimum inhibitory concentration of the one or morecarbapenem in the range of 2 to 32 fold; that is sufficient to reducethe minimum inhibitory concentration of the one or more carbapenems bygreater or equal to 2-fold; that is sufficient to reduce the minimuminhibitory concentration of the one or more carbapenems by greater orequal to 3-fold; that is sufficient to reduce the minimum inhibitoryconcentration of one or more carbapenems by greater or equal to 5-fold;that is sufficient to reduce the minimum inhibitory concentration of oneor more carbapenems by greater or equal to 8-fold; that is sufficient toreduce the minimum inhibitory concentration of one or more carbapenemsby greater or equal to 13-fold; or that is sufficient to reduce theminimum inhibitory concentration of one or more carbapenems by greateror equal to 38-fold and bring it to therapeutically usable doses.Alternatively, the one or more carbapenem is selected from the groupconsisting of tebipenem, ertapenem, and panipenem.

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

By “agent” is meant any small molecule chemical compound, antibody,nucleic acid molecule, or polypeptide, or fragments thereof.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease.

By “analog” is meant a molecule that is not identical, but has analogousfunctional or structural features.

By “disease” is meant any condition or disorder that damages orinterferes with the normal function of a cell, tissue, or organ.Examples of diseases include a bacterial infection.

By “effective amount” is meant the amount of a required to amelioratethe symptoms of a disease relative to an untreated patient. Theeffective amount of active compound(s) used to practice the presentinvention for therapeutic treatment of a disease varies depending uponthe manner of administration, the age, body weight, and general healthof the subject. Ultimately, the attending physician or veterinarian willdecide the appropriate amount and dosage regimen. Such amount isreferred to as an “effective” amount.

By “reduces” is meant a negative alteration of at least 10%, 25%, 50%,75%, or 100%.

A “reference” refers to a standard or control conditions such as asample (human cells) or a subject that is a free, or substantially free,of an agent such as one or more agents of the present inventioncomprising a β-lactamase inhibitor and a carbapenem.

As used herein, the term “subject” is intended to refer to anyindividual or patient to which the method described herein is performed.Generally the subject is human, although as will be appreciated by thosein the art, the subject may be an animal. Thus other animals, includingmammals such as rodents (including mice, rats, hamsters and guineapigs), cats, dogs, rabbits, farm animals including cows, horses, goats,sheep, pigs, etc., and primates (including monkeys, chimpanzees,orangutans and gorillas) are included within the definition of subject.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a”, “an”, and “the” areunderstood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

As used herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment” and the like refer to reducing the probabilityof developing a disorder or condition in a subject, who does not have,but is at risk of or susceptible to developing a disorder or condition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the distribution of susceptibility of M. abscessusstrains (n=28) against carbapenems with or without 4 μg/mL avibactam(Avi). The data labels in each plot represent the number of isolates.Tebipenem (Tebi), ertapenem (Erta), panipenem (Pani), doripenem (Dori),biapenem (Bia), meropenem (Mero), faropenem (Faro) and imipenem (Imi).

FIG. 2 illustrates Table 2: Antimicrobial susceptibility profiles oftwenty eight M. abscessus clinical strains against carbapenems (with andwithout avibactam, 4 μg/mL) and antibacterials that are currently usedto treat M. abscessus infection and where available currently used CLSIbreakpoints for interpretation. CLR, clarithromycin (susceptible, 0.06μg/mL to 2 μg/mL, intermediate, 4 μg/mL, resistant, ≥8 μg/mL); SXT,trimethoprim/sulfamethoxazole (susceptible, 0.25/4.75 to 2/38 μg/mL,resistant, ≥4/76 μg/mL); CIP, ciprofloxacin (susceptible, 0.12 μg/mL to1 μg/mL, intermediate, 2 μg/mL, resistant, ≥4 μg/mL); MXF, moxifloxacin(susceptible, 0.25 μg/mL to 1 μg/mL, intermediate, 2 μg/mL, resistant,≥4 μg/mL); FOX, cefoxitin (susceptible, 4 μg/mL to 16 μg/mL,intermediate, 32-64 μg/mL, resistant, ≥128 μg/mL); AMI, amikacin(susceptible, 1 μg/mL to 16 μg/mL, intermediate, 32 μg/mL, resistant,≥64 μg/mL); TGC, tigecycline (no interpretation for this drug currentlyavailable, however an MIC>4 μg/mL for M. tuberculosis is consideredresistant in broth); LZD, linezolid (susceptible, 1 μg/mL to 8 μg/mL,intermediate, 16 μg/mL, resistant, ≥32 μg/mL); IMI, imipenem(susceptible, 2 μg/mL to 4 μg/mL, intermediate, 8 μg/mL to 16 μg/mL,resistant, ≥32 μg/mL); TOB, tobramycin (susceptible, 1 μg/mL to 2 μg/mL,intermediate, 4 μg/mL, resistant, ≥8 μg/mL); KAN, kanamycin (nointerpretation for this drug currently available, however, an MIC>5μg/ml for M. tuberculosis is considered resistant in broth; Tebipenem(Tebi), ertapenem (Erta), panipenem (Pani), doripenem (Dori), biapenem(Bia), meropenem (Mero), faropenem (Faro), imipenem (Imi), avibactam(Avi) and not determined (ND).

DETAILED DESCRIPTION OF THE INVENTION

The peptidoglycan is an Achilles's heel of bacteria as agents thatinhibit its biosynthesis, namely β-lactams and glycopeptides, comprisesome of the most widely used class of antibacterials in modem medicine.β-lactams derive their activity by preventing formation of linkagebetween peptide sidechains by inhibiting the transpeptidases thatcatalyze this reaction. Recently it was demonstrated that majority ofthe linkages in the peptidoglycan layer of M. abscessus are generated byLD-transpeptidases and that this class of enzyme is selectively moresusceptible to the carbapenem class of β-lactams. Imipenem, acarbapenem, has superior activity compared to cefoxitin against clinicalstrains of M. abscessus isolated from cystic fibrosis patients. M.abscessus harbors a chromosomally encoded β-lactamase that is highlyactive and therefore is of major concern while considering β-lactams fortreatment of M. abscessus infections. Here, we have studied ifavibactam, a recently developed β-lactamase inhibitor, can alter thepotency of the carbapenem class of β-lactams against M. abscessus.Recent studies have provided insight into the activities of some oldercarbpenems with or without avibactam against M. abscessus. We haveincluded all commercially available carbapenems, most importantly newand oral carbapenems, and a collection of clinically isolated M.abscessus strains most of which are resistant to multiple drugscurrently deployed to treat infection by this pathogen. Activities ofthe combinations of clavulanate, a β-lactamase inhibitor, andcarbapenems, were recently reported; therefore clavulanate was excludedfrom this study.

Bacterial Strains

Twenty eight unique clinical isolates of M. abscessus were used in thisstudy. These strains were obtained de-identified from the archive of theclinical microbiology laboratory of the Johns Hopkins UniversityHospital per institutional ethical guidelines. They were isolated over a10 year period, from 2005-2015, from patients that were temporally andgeographically unrelated. No two isolates are from the same patient.Those displaying a high level of resistance to antibacterials used forM. abscessus infection were selected for this study. All strainsobtained prior to 2014 were identified to the M. abscessus complex levelusing a variety of methods including 16S rDNA sequencing in conjunctionwith selected biochemical testing such as sodium citrate. More recentisolates (those isolated after 2014) were identified using MALDI ToFmass spectrometry in which a Bruker MicroFlex LT (MicroFlex LT) massspectrometer and Bruker Biotyper software and existing database (version2.0, Bruker) were employed. Sub-speciation within the M. abscessuscomplex, which helps to distinguish between M. abscessus sensu stricto,M. massiliense, and M. bolletii, was not done as this is not per thecurrent standard of care at the Johns Hopkins Clinical MycobacteriologyLaboratory. Distinguishing between M. abscessus sensu stricto and M.massiliense is most often performed to guide therapy since M.massiliense is known to have a non-functional erm gene and is thereforesusceptible to macrolides. However, due to the high number of macrolideresistant M. abscessus complex isolates recovered at Johns Hopkins, mostpatient isolates are subjected to drug susceptibility testing, makingspeciation within the complex of lesser importance. Thus, the proportionof each subspecies within the M. abscessus complex for the Johns Hopkinsstrain collection is not known. M. abscessus ATCC 19977 was included asa reference drug-sensitive strain.

Growth Conditions and Minimum Inhibitory Concentration (MIC)

All strains were initially grown in 7H9 complete medium composed ofMiddlebrook 7H9 broth (Difco) supplemented with 0.5% glycerol, 10% oleicacid-albumin-dextrose-catalase and 0.05% Tween-80 at 37° C. withconstant shaking. A standard broth microdilution method [20] was used todetermine MIC. Briefly, M. abscessus strains were grown as describedabove and these cultures, at exponential phase (A_(600 nm)˜0.6-0.8),were used to inoculate 10⁵ colony forming units (CFU) into each well ofmicrotiter culture plates containing a carbapenem at two fold serialdilutions ranging from 256 to 0.25 μg/mL. An identical setup but withwells containing 4 μg/mL of avibactam was used to assess the effect ofthis agent on each carbapenem. Carbapenems studied were procuredcommercially from Sigma-Aldrich and include ertapenem, meropenem,imipenem, doripenem, biapenem, faropenem, tebipenem and panipenem.β-lactamase inhibitors sulbactam, tazobactam and avibactam were alsoprocured from Sigma-Aldrich. In addition, to establish baselineantibacterial susceptibility for each strain we also determined MICs fordrugs currently used to treat M. abscessus infections. They arelinezolid, cefoxitin, kanamycin, ciprofloxacin, moxifloxacin, amikacin,tigecycline, imipenem, tobramycin, trimethoprim/sulfamethoxazole, andclarithromycin. M. abscessus growth was evaluated by visual inspectionof pellets after 3 days of incubation (the exception beingclarithromycin for which incubation was extended to 14 days) at 30° C.without shaking per CLSI guidelines [21]. In addition to assessment in7H9 complete medium, we also used cation adjusted Mueller-Hinton brothfor MIC studies for the reference strain M. abscessus ATCC 19977. Allexperiments were repeated and the final data represent the average oftwo biological replicates.

We studied the utility of β-lactamase inhibitors sulbactam, tazobactam,and avibactam in restoring in vitro potencies of carbapenems against M.abscessus using the reference drug-susceptible strain ATCC 19977. Wedetermined MICs of ertapenem, meropenem, imipenem, doripenem, biapenem,faropenem, tebipenem and panipenem alone or in the presence of theaforementioned four β-lactamase inhibitors. In general, addition ofsulbactam and tazobactam failed to reduce the MICs of the carbapenems(Table 1). However, avibactam (4 μg/mL) consistently reduced the MICs ofcarbapenems by 2 to 32 fold. Based on these data, we selected avibactamfor further consideration.

TABLE 1 Minimum inhibitory concentrations (in μg/mL) of carbapenems withand without β-lactamase inhibitors against M. abscessus ATCC 19977.β-lactamase inhibitors sulbactam, tazobactam and avibactam were used ata fixed concentration of 4 μg/mL. Cation-adjusted Mueller-Hinton broth(CAMHB), not determined (ND). 7H9 7H9 + 7H9 + 7H9 + CAMHB CAMHB + Drugbroth sulbactam tazobactam avibactam only avibactam Ertapenem 64-128 >6432-64  4-8 128-256  8-16 Meropenem 8-16 8-16 8-16 2-4 32-64 4-8 Imipenem4-8  4-8  2-4  2-4  8-16 4-8 Doripenem 8-16 8-16 8-16 2-4 16-32 4-8Biapenem 8-16 4-8  8-16 2-4 16-32  8-16 Faropenem 32-64  16-32  32-64  8-16  64-128 16-32 Tebipenem 128-256  >64 >64 4-8 128-256  8-16Panipenem 64-128 16-32  32-64   8-16  64-128  8-16 Sulbactam >64 ND NDND ND ND Tazobactam >64 ND ND ND ND ND Avibactam >256  ND ND ND >256 ND

The twenty eight clinical isolates included in this study exhibited awide range of susceptibilities and resistance to the antibacterials usedto treat M. abscessus infections (FIG. 2 of Table 2). This baselineprofile illustrates that these clinical isolates are mostly resistant tothe drugs that are part of the current recommendation for treatment ofM. abscessus infections, with tigecycline being the only exception. TheMIC of carbapenems against the drug sensitive control strain M.abscessus ATCC 19977 were as expected. They were 128, 16, 8, 16, 16, 64,256 and 128 μg/mL for ertapenem, meropenem, imipenem, doripenem,biapenem, faropenem, tebipenem and panipenem, respectively. Brothmicrodilution breakpoints for imipenem and meropenem have beenestablished for rapidly-growing mycobacteria, including M. abscessus andare the same for both drugs: susceptible, ≤4 μg/mL, intermediate 8 μg/mLto 16 μg/mL, and resistant ≥32 μg/mL. However, breakpoints for M.abscessus have not been established for any of the other carbapenemstested in this study. In such cases, comparison of individual MICs toaverage peak plasma level and half-life may be useful clinically(ertapenem: average peak plasma concentration ˜150 μg/mL with ahalf-life of ˜4 hrs; tebipenem: average peak plasma concentration ˜8μg/mL with a half-life of 1 hr; panipenem: average peak plasmaconcentration ˜22 μg/mL with a half-life of 40 to 70 minutes).

Compared to ATCC 19977, the twenty eight clinical isolates were equallyor more resistant to the carbapenems with a few exceptions (FIG. 2 ofTable 2). The average reduction in MICs of the carbapenems, incombination with avibactam, against the twenty eight clinical strains isas follows: imipenem (2 fold), faropenem (3 fold), biapenem (5 fold),doripenem (6 fold), meropenem (6 fold), panipenem (8 fold), ertapenem(>13 fold) and tebipenem (>38 fold) (FIG. 1). While avibactam was leasteffective in reducing the MICs of imipenem and faropenem against M.abscessus it was able to restore the MICs of panipenem, ertapenem andtebipenem to levels that are therapeutically relevant. These data alsoshow an interesting pattern: irrespective of how high or low the MIC ofa carbapenem alone, avibactam can reduce the final MIC to no lower than4-8 μg/mL. Therefore, it appears that there is a lower limit for MIC ofcarbapenem and avibactam combination.

Discussion

M. abscessus is considered to be the most virulent of the rapidlygrowing mycobacteria. This organism most commonly affectsimmunocompromised hosts; for example, those with cystic fibrosis andlung transplant recipients, and is largely considered to be a chronicand incurable disease. Effective antimicrobial treatment is sometimesthe only thing that stands between these patients and overwhelminginfection or even death.

The treatment regimen for M. abscessus pulmonary disease generallyinvolves combination therapy with multiple agents for an extended courseof several months. Many of these antibiotics are poorly-tolerated andare associated with significant cytotoxic effects. The high prevalenceof both intrinsic and acquired antimicrobial resistance furthercomplicates this regimen, making development of novel treatmentstrategies crucial. M. abscessus genome encodes a β-lactamase,Bla_(Mab), which is not effectively inhibited by clavulanate, sulbactamand tazobactam. We have also failed to observe significant reduction inMIC of carbapenems against M. abscessus when supplemented withclavulanate or sulbactam or tazobactam (Table 1). Bla_(Mab) has beenshown to hydrolyze β-lactams with high efficiency, especially imipenem.While this study suggested that avibactam could potentially protectimipenem from Bla_(Mab), in our study, the MIC of imipenem was leastaltered by avibactam (FIG. 1 and FIG. 2 of Table 2). Therefore, itappears that M. abscessus likely possess an additional way to degradeimipenem even when it is protected from Bla_(Mab) by avibactam. On theother hand, it is plausible that tebipenem, ertapenem and panipenem areefficiently hydrolyzed by Bla_(Mab) and any additional mechanism presentin M. abscessus, and avibactam effectively protects these carbapenems.

Our study showed that the addition of avibactam to various carbapenemantibiotics effectively reduced the MICs of carbapenem-resistant M.abscessus isolates to within therapeutically-achievable levels in vitro.Additionally, the largest reduction in MIC was achieved with tebipenem,which is available in an oral formulation and may further simplify themulti-drug treatment regimen.

Both carbapenems and β-lactamase inhibitors are FDA approved, widelyavailable, and generally well-tolerated. The results of our study arehighly promising, as they denote a potential new treatment strategy forcarbapenem-resistant M. abscessus that could be easily implemented inclinical practice.

Avibactam is the most active β-lactamase inhibitor in reducing the MICsof tebipenem, ertapenem and panipenem; if it were not for avibactam, theMIC of these carbapenems would be well outside the clinically relevanttherapeutic window. For those carbapenems that exhibit high MIC againstM. abscessus, addition of avibactam usually reduces the MIC. However, itappears that avibactam reduces the MIC to 2-8 μg/mL, but not below thisrange, irrespective of how high the MIC of a carbapenem alone is.

The present invention determined that avibactam can restore potency ofcarbapenems against M. abscessus. Using a full panel of carbapenemsagainst a collection of independent M. abscessus clinical isolates, theinventors observed that for select carbapenems, avibactam greatlyreduces their MIC. Avibactam reduces the MIC of tebipenem against M.abscessus to 4-8 μg/mL, representing a 32-64 fold decrease. Thisconcentration is achievable in the blood making tebipenem-avibactamcombination a potentially new regimen for treatment of M. abscessusinfection. Avibactam also reduces the MIC of ertapenem and panipenemagainst M. abscessus to levels achievable in the blood.

Embodiments of the disclosure concern methods and/or compositions fortreating and/or preventing a bacterial infection. In certainembodiments, individuals with a bacterial infection are treated with anagent comprising a β-lactamase inhibitor and a carbapenem of the presentinvention wherein the β-lactamase inhibitor reduces the minimuminhibitory concentration of one or more carbpenem in the range of 2 to32 fold, or any of the ranges described in this specification.

In certain embodiments, the level to which a β-lactamase inhibitorreduces the minimum inhibitory concentration of one or more carbpenemmay be any level so long as it provides amelioration of at least onesymptom of the bacterial infection. The β-lactamase inhibitor may reducethe minimum inhibitory concentration of one or more carbpenem by atleast 2, 3, 4, 5, 10, 25, of 50, fold compared to the level ofexpression in a standard (not provided the β-lactamase inhibitor), in atleast some cases.

In particular embodiments of the disclosure, an individual is given anagent for a bacterial invention in addition to the one or more agents ofthe present invention including a β-lactamase inhibitor and acarbapenem. Such additional therapy may include antibiotics. Whencombination therapy is employed with an agent of the present invention,the additional therapy may be given prior to, at the same time as,and/or subsequent to the agent of the present invention.

Pharmaceutical Preparations

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more agents of the present invention, such asa β-lactamase inhibitor and a carbapenem, dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of a pharmaceutical composition thatcomprises at least one agent of the present invention or additionalactive ingredient will be known to those of skill in the art in light ofthe present disclosure, as exemplified by Remington: The Science andPractice of Pharmacy, 21^(st) Ed. Lippincott Williams and Wilkins, 2005,incorporated herein by reference. Moreover, for animal (e.g., human)administration, it will be understood that preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the pharmaceuticalcompositions is contemplated.

The antibacterial agents of the present invention may comprise differenttypes of carriers depending on whether it is to be administered insolid, liquid or aerosol form, and whether it need to be sterile forsuch routes of administration as injection. The present compositions canbe administered intravenously, intradermally, transdermally,intrathecally, intraarterially, intraperitoneally, intranasally,intravaginally, intrarectally, topically, intramuscularly,subcutaneously, mucosally, orally, topically, locally, inhalation (e.g.,aerosol inhalation), injection, infusion, continuous infusion, localizedperfusion bathing target cells directly, via a catheter, via a lavage,in cremes, in lipid compositions (e.g., liposomes), or by other methodor any combination of the forgoing as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, incorporated herein by reference).The antibacterial agents of the present invention may be provided to theindividual in need thereof by dietary ingesting one or more comestiblesthat comprise the inducer, such as herbs, berries, and/or fruits.

The agents of the present invention including a β-lactamase inhibitorand a carbapenem described in this invention may be formulated into acomposition in a free base, neutral or salt form. Pharmaceuticallyacceptable salts, include the acid addition salts, e.g., those formedwith the free amino groups of a proteinaceous composition, or which areformed with inorganic acids such as for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric ormandelic acid. Salts formed with the free carboxyl groups can also bederived from inorganic bases such as for example, sodium, potassium,ammonium, calcium or ferric hydroxides; or such organic bases asisopropylamine, trimethylamine, histidine or procaine. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas formulated for parenteral administrations such as injectablesolutions, or aerosols for delivery to the lungs, or formulated foralimentary administrations such as drug release capsules and the like.

Further in accordance with the present disclosure, the composition ofthe present invention, including the agents and pharmaceuticalcompositions including the agents, suitable for administration isprovided in a pharmaceutically acceptable carrier with or without aninert diluent. The carrier should be assimilable and includes liquid,semi-solid, i.e., pastes, or solid carriers. Except insofar as anyconventional media, agent, diluent or carrier is detrimental to therecipient or to the therapeutic effectiveness of a composition containedtherein, its use in administrable composition for use in practicing themethods of the present invention is appropriate. Examples of carriers ordiluents include fats, oils, water, saline solutions, lipids, liposomes,resins, binders, fillers and the like, or combinations thereof. Thecomposition may also comprise various antioxidants to retard oxidationof one or more component. Additionally, the prevention of the action ofmicroorganisms can be brought about by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol,sorbic acid, thimerosal or combinations thereof.

In accordance with the present invention, the composition is combinedwith the carrier in any convenient and practical manner, i.e., bysolution, suspension, emulsification, admixture, encapsulation,absorption and the like. Such procedures are routine for those skilledin the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in an thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle compositions that include one or moreantibacterial agent of the present invention, one or more lipids, and anaqueous solvent. As used herein, the term “lipid” will be defined toinclude any of a broad range of substances that is characteristicallyinsoluble in water and extractable with an organic solvent. This broadclass of compounds are well known to those of skill in the art, and asthe term “lipid” is used herein, it is not limited to any particularstructure. Examples include compounds which contain long-chain aliphatichydrocarbons and their derivatives. A lipid may be naturally occurringor synthetic (i.e., designed or produced by man). However, a lipid isusually a biological substance. Biological lipids are well known in theart, and include for example, neutral fats, phospholipids,phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids,glycolipids, sulphatides, lipids with ether and ester-linked fatty acidsand polymerizable lipids, and combinations thereof. Of course, compoundsother than those specifically described herein that are understood byone of skill in the art as lipids are also encompassed by thecompositions and methods of the present invention.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the inducer of expression of PGC-1

dissolved with a lipid, emulsified with a lipid, mixed with a lipid,combined with a lipid, covalently bonded to a lipid, contained as asuspension in a lipid, contained or complexed with a micelle orliposome, or otherwise associated with a lipid or lipid structure by anymeans known to those of ordinary skill in the art. The dispersion may ormay not result in the formation of liposomes.

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration.

Depending upon the dosage and the route of administration, the number ofadministrations of a preferred dosage and/or an effective amount mayvary according to the response of the subject. The practitionerresponsible for administration will, in any event, determine theconcentration of active ingredient(s) in a composition and appropriatedose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

Alimentary Compositions and Formulations

In one embodiment of the present disclosure, the antibacterial agents ofthe present invention are formulated to be administered via analimentary route. Alimentary routes include all possible routes ofadministration in which the composition is in direct contact with thealimentary tract. Specifically, the pharmaceutical compositionsdisclosed herein may be administered orally, buccally, rectally, orsublingually. As such, these compositions may be formulated with aninert diluent or with an assimilable edible carrier, or they may beenclosed in hard- or soft-shell gelatin capsule, or they may becompressed into tablets, or they may be incorporated directly with thefood of the diet.

In certain embodiments, the active compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tables,troches, capsules, elixirs, suspensions, syrups, wafers, and the like(Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515;5,580,579 and 5,792, 451, each specifically incorporated herein byreference in its entirety). The tablets, troches, pills, capsules andthe like may also contain the following: a binder, such as, for example,gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; anexcipient, such as, for example, dicalcium phosphate, mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate or combinations thereof; a disintegrating agent, such as, forexample, corn starch, potato starch, alginic acid or combinationsthereof; a lubricant, such as, for example, magnesium stearate; asweetening agent, such as, for example, sucrose, lactose, saccharin orcombinations thereof; a flavoring agent, such as, for examplepeppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.When the dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both. When the dosage form is a capsule, it maycontain, in addition to materials of the above type, carriers such as aliquid carrier. Gelatin capsules, tablets, or pills may be entericallycoated. Enteric coatings prevent denaturation of the composition in thestomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No.5,629,001. Upon reaching the small intestines, the basic pH thereindissolves the coating and permits the composition to be released andabsorbed by specialized cells, e.g., epithelial enterocytes and Peyer'spatch M cells. A syrup of elixir may contain the active compound sucroseas a sweetening agent methyl and propylparabens as preservatives, a dyeand flavoring, such as cherry or orange flavor. Of course, any materialused in preparing any dosage unit form should be pharmaceutically pureand substantially non-toxic in the amounts employed. In addition, theactive compounds may be incorporated into sustained-release preparationand formulations.

For oral administration the compositions of the present disclosure mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

Additional formulations which are suitable for other modes of alimentaryadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum. After insertion, suppositories soften, melt or dissolvein the cavity fluids. In general, for suppositories, traditionalcarriers may include, for example, polyalkylene glycols, triglyceridesor combinations thereof. In certain embodiments, suppositories may beformed from mixtures containing, for example, the active ingredient inthe range of about 0.5% to about 10%, and preferably about 1% to about2%.

Parenteral Compositions and Formulations

In further embodiments, antibacterial agents of the present inventionmay be administered via a parenteral route. As used herein, the term“parenteral” includes routes that bypass the alimentary tract.Specifically, the pharmaceutical compositions disclosed herein may beadministered for example, but not limited to intravenously,intradermally, intramuscularly, intraarterially, intrathecally,subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,7537,514, 6,613,308,5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each specificallyincorporated herein by reference in its entirety).

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy injectability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (i.e., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in isotonic NaCl solution andeither added hypodermoclysis fluid or injected at the proposed site ofinfusion, (see for example, “Remington's Pharmaceutical Sciences” 15thEdition, pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. A powdered composition is combined with a liquidcarrier such as, e.g., water or a saline solution, with or without astabilizing agent.

Miscellaneous Pharmaceutical Compositions and Formulations

In other preferred embodiments of the invention, the active compound orantibacterial agents of the present invention may be formulated foradministration via various miscellaneous routes, for example, topical(i.e., transdermal) administration, mucosal administration (intranasal,vaginal, etc.) and/or inhalation.

Pharmaceutical compositions for topical administration may include theactive compound formulated for a medicated application such as anointment, paste, cream or powder. Ointments include all oleaginous,adsorption, emulsion and water-solubly based compositions for topicalapplication, while creams and lotions are those compositions thatinclude an emulsion base only. Topically administered medications maycontain a penetration enhancer to facilitate adsorption of the activeingredients through the skin. Suitable penetration enhancers includeglycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones andluarocapram. Possible bases for compositions for topical applicationinclude polyethylene glycol, lanolin, cold cream and petrolatum as wellas any other suitable absorption, emulsion or water-soluble ointmentbase. Topical preparations may also include emulsifiers, gelling agents,and antimicrobial preservatives as necessary to preserve the activeingredient and provide for a homogenous mixture. Transdermaladministration of the present invention may also comprise the use of a“patch”. For example, the patch may supply one or more active substancesat a predetermined rate and in a continuous manner over a fixed periodof time.

In certain embodiments, the pharmaceutical compositions may be deliveredby eye drops, intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering compositions directly to thelungs via nasal aerosol sprays has been described e.g., in U.S. Pat.Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein byreference in its entirety). Likewise, the delivery of drugs usingintranasal microparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts. Likewise, transmucosal drugdelivery in the form of a polytetrafluoroetheylene support matrix isdescribed in U.S. Pat. No. 5,780,045 (specifically incorporated hereinby reference in its entirety).

The term aerosol refers to a colloidal system of finely divided solid ofliquid particles dispersed in a liquefied or pressurized gas propellant.The typical aerosol of the present invention for inhalation will consistof a suspension of active ingredients in liquid propellant or a mixtureof liquid propellant and a suitable solvent. Suitable propellantsinclude hydrocarbons and hydrocarbon ethers. Suitable containers willvary according to the pressure requirements of the propellant.Administration of the aerosol will vary according to subject's age,weight and the severity and response of the symptoms.

Kits of the Disclosure

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, an antibacterial agent (for example, a β-lactamaseinhibitor and a carbapenem of the present invention) may be comprised ina kit.

The kits may comprise a suitably aliquoted of an antibacterial agent ofthe present invention and, in some cases, one or more additional agents.The component(s) of the kits may be packaged either in aqueous media orin lyophilized form. The container means of the kits will generallyinclude at least one vial, test tube, flask, bottle, syringe or othercontainer means, into which a component may be placed, and preferably,suitably aliquoted. Where there are more than one component in the kit,the kit also will generally contain a second, third or other additionalcontainer into which the additional components may be separately placed.However, various combinations of components may be comprised in a vial.The kits of the present invention also will typically include a meansfor containing the bacterial agent of the present invention and anyother reagent containers in close confinement for commercial sale. Suchcontainers may include injection or blow-molded plastic containers intowhich the desired vials are retained.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. The antibacterial agentsof the present invention and composition(s) thereof may be formulatedinto a syringeable composition. In which case, the container means mayitself be a syringe, pipette, and/or other such like apparatus, fromwhich the formulation may be applied to an infected area of the body,injected into an animal, and/or even applied to and/or mixed with theother components of the kit.

However, the components of the kit may be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent. It isenvisioned that the solvent may also be provided in another containermeans.

Examples of Chemical Structures

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method of treating or preventing a bacterial infection comprisingthe following steps: administering an agent comprising a β-lactamaseinhibitor and one or more carbapenem to a subject and treating orpreventing a bacterial infection in the subject.
 2. The method of claim1 wherein the carbapenem is selected from the group consisting ofertapenem, meropenem, imipenem, doripenem, biapenem, faropenem,tebipenem, panipenem and a combination thereof.
 3. The method of claim 1wherein the β-lactamase inhibitor is selected from the group consistingof avibactam or clavulanate, or a combination thereof.
 4. The method ofclaim 1 wherein the treating of the bacteria infection occurs when atleast one symptom of the bacteria infection is alleviated compared to areference subject that was not been administered the agent.
 5. Themethod of claim 1 wherein the β-lactamase inhibitor is avibactam.
 6. Themethod of claim 1 wherein the bacterial infection comprisesMycobacterium abscessus.
 7. The method of claim 1 wherein the amount ofβ-lactamase inhibitor is sufficient of to reduce the minimum inhibitoryconcentration of the one or more carbapenem in the range of 2 to 32fold.
 8. The method of claim 1 wherein the amount of β-lactamaseinhibitor is sufficient to reduce the minimum inhibitory concentrationof the one or more carbapenems by greater or equal to 2-fold.
 9. Themethod of claim 1 wherein the amount of β-lactamase inhibitor issufficient to reduce the minimum inhibitory concentration of the one ormore carbapenems by greater or equal to 3-fold.
 10. The method of claim1 wherein the amount of β-lactamase inhibitor is sufficient to reducethe minimum inhibitory concentration of one or more carbapenems bygreater or equal to 5-fold.
 11. The method of claim 1 wherein the amountof β-lactamase inhibitor is sufficient to reduce the minimum inhibitoryconcentration of one or more carbapenems by greater or equal to 8-fold.12. The method of claim 1 wherein the amount of β-lactamase inhibitor issufficient to reduce the minimum inhibitory concentration of one or morecarbapenems by greater or equal to 13-fold.
 13. The method of claim 1wherein the amount of β-lactamase inhibitor is sufficient to reduce theminimum inhibitory concentration of one or more carbapenems by greateror equal to 38-fold.
 14. The method of claim 1 wherein the one or morecarbapenem is selected from the group consisting of tebipenem,ertapenem, and panipenem.
 15. The method of claim 1 wherein the one ormore carbapenem is tebipenem.